Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using bacteria
Reexamination Certificate
2001-10-23
2004-03-16
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using bacteria
C435S252300, C435S440000, C435S441000, C435S233000, C435S234000, C435S195000, C435S183000, C435S173800, C435S471000, C435S479000, C435S481000, C435S482000, C435S069100
Reexamination Certificate
active
06706503
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for directing the evolution of microorganisms using mutator genes. Such methods provide a pool of microbial genetic diversity advantageous for industrial application, such as for the industrial production of heterologous proteins, such as hormones, growth factors and enzymes, and the biocatalytic production of chemicals, vitamins, amino acids and dyes.
BACKGROUND OF THE INVENTION
The industrial applicability of microorganisms is restricted by their physiological limits set by solvent, pH, various solutes, salts and temperature. Organic solvents are generally toxic to microorganisms even at low concentrations. The toxicity of solvents significantly limits use of microorganisms in industrial biotechnology for production of specialty chemicals and for bioremediation applications. Solvent molecules incorporate into bacterial membranes and disrupt membrane structure (Isken and Bont, 1998
, Extremophiles
2(3): 229-238); (Pinkart and White, 1997
, J. Bacteriol.
179(13): 4219-4226); (Ramos, Duque et al., 1997
, “J. Biol. Chem
. 272(7): 3887-3890); (Alexandre, Rousseaux et al., 1994
, FEMS Microbiol, Lett,
124(1): 17-22); and Kieboom, Dennis et al., 1998
, J. of Bacteriology
180(24): 6769-6772). Classic strain improvement methods including UV and chemical mutagenesis have been applied for selection of more tolerant strains (Miller, J., “A Short Course In Bacterial Genetics,” Cold Spring Harbor Laboratory Press, 1992). A number of studies have been dedicated to identification and isolation of solvent tolerant mutants among various bacterial strains. Spontaneous
E. coli
solvent tolerant mutants and mutants isolated in the process of 1-methyl-3-nitrosoguanidine (NTG) mutagenesis were obtained from strain K-12 (Aono, Albe et al., 1991
Agric. Biol. Chem
55(7): 1935-1938). The mutants could grow in the presence of diphenylether, n-hexane, propylbenzene, cyclohexane, n-pentane, p-xylene. Various Pseudomonas strains were able to adapt and to grow in a toluene-water two-phase system (Inoue and Horikoshi, 1989
, Nature
338: 264-266), with p-xylene (Cruden, Wolfram et al., 1992
, Appl. Environ. Microbiol
. 58(9): 2723-2729), styrene and other organic solvents (Weber, Ooijkaas et al., 1993
, Appl. Environ. Microbiol
. 59(10): 3502-3504), (de Bont 1998
, Trends in Biotechnology
16: 493-499). Yomano et al. isolated ethanol tolerant mutants which increased tolerance from 35 g/l to 50 g/l during 32 consequent transfers (Yomano, York et al., 1998
, J. Ind. Microbiol. Biotechnol
. 20(2): 132-138). High temperature evolution using
E. coli
has been disclosed in the art (Bennett, 1990
, Nature
, Vol. 346, 79-81) however the fitness gain was low as compared to the parent.
Strains of
E. coli
that carry mutations in one of the DNA repair pathways have been described which have a higher random mutation rate than that of typical wild type strains (see, Miller supra, pp. 193-211). As reported by Degenen and Cox (
J. Bacteriol.,
1974, Vol. 117, No. 2, pp.477-487), an
E. coli
strain carrying a mutD5 allele demonstrates from 100 to 10,000 times the mutation rate of its wild type parent. Greener et al., “Strategies In Molecular Biology,” 1994, Vol. 7, pp.32-34, disclosed a mutator strain that produces on average one mutation per 2000 bp after growth for about 30 doublings.
Microorganisms are used industrially to produce desired proteins, such as hormones, growth factors and enzymes and to produce chemicals, such as glycerol and 1,3 propanediol (WO 98/21340 published May 22, 1998 and U.S. Pat. No. 5,686,276 issued Nov. 11, 1997), vitamins, such as ascorbic acid intermediates (1985
, Science
230:144-149), amino acids, and dyes, such as indigo (U.S. Pat. No. 4,520,103, issued May 28, 1985). In spite of advances in the art, there remains a need to improve the microorganisms and methods for producing such desired proteins, chemicals, amino acids and dyes.
SUMMARY OF THE INVENTION
The present invention relates generally to methods for directing the evolution of a microoganism, that is for directing desired genetic change in a microorganism in response to conditions of selective pressure. In one aspect, the present invention relates to methods for evolving microorganisms to grow under extreme conditions, such as at high temperature, under conditions of pH extremes, in the presence of solvents, and in the presence of high salt. In another aspect, the present invention relates to methods for evolving a microorganism comprising at least one nucleic acid encoding a desired protein or an enzyme in an enzymatic pathway to grow under desired conditions.
The present invention is based, in part, upon the finding that microrganisms such as wild-type
E. coli
and
E. blattae
, can be evolved into microorganisms capable of growing in the presence of high solvents, such as DMF and 1,3 propanediol, using methods described herein. The present invention is also based, in part, upon the finding that
E. coli
can be evolved into a microorganism capable of growing at elevated temperatures using methods described herein. The present invention is further based, in part, upon the identification of the optimal mutation rate for a microorganism and the discovery that the mutation rate can be controlled.
Accordingly, the present invention provides a method for preparing an evolved microorganism comprising the steps of culturing a microorganism comprising at least one heterologous mutator gene for at least 20 doublings under conditions suitable for selection of an evolved microorganism, wherein said heterologous mutator gene generates a mutation rate of at least about 5 fold to about 100,000 fold relative to wild type, and restoring said evolved microorganism to a wild type mutation rate. In one embodiment, the microorganism further comprises at least one introduced nucleic acid encoding a heterologous protein, said protein(s) including, but not limited to hormones, enzymes, growth factors. In another embodiment, the enzyme includes, but is not limited to hydrolases, such as protease, esterase, lipase, phenol oxidase, permease, amylase, pullulanase, cellulase, glucose isomerase, laccase and protein disulfide isomerase. The present invention encompasses genetic changes in the microorganism as well as changes in the introduced nucleic acid.
In yet a further embodiment, the microorganism further comprises introduced nucleic acid encoding at least one enzyme necessary for an enzymatic pathway. In one embodiment, the introduced nucleic acid is heterologous to the microorganism; in another, the introduced nucleic acid is homologous to the microorganism. In a further embodiment, the enzyme is a reductase or a dehydrogenase and said enzymatic pathway is for the production of ascorbic acid or ascorbic acid intermediates. In an additional embodiment, the enzyme is glycerol dehydratase or 1,3-propanediol dehydrogenase and said enzymatic pathway is for the production of 1,3 propanediol, 1,3 propanediol precursors or 1,3 propanediol derivatives. In another embodiment, the enzyme is glycerol-3-phosphate dehydrogenase or glycerol-3-phosphate phosphatase and said pathway is for the production of glycerol and glycerol derivatives. In a further embodiment, the enzymatic pathway is for the production of amino acids, such as tryptophane or lysine or dyes, such as indigo.
In one embodiment of the present invention, the microorganism is cultured for between about 20 to about 100 doublings; in another embodiment, the microorganism is cultured for between about 100 to about 500 doublings; in yet another embodiment, the microorganism is cultured for between about 500 to about 2000 doublings and in a further embodiment, the microorganism is cultured for greater than 2000 doublings. In one embodiment, the mutator gene generates a mutation rate of at least about 5 fold to about 10,000 fold relative to wild type; in another embodiment, the mutator gene generates a mutation rate of a least about 5 fold to about 1000 fold and in another embodiment, the mutator gene generates a mutation rate of a
Liu Amy D.
Schellenberger Volker
Selifonova Olga V.
Genencor International Inc.
Genencor International, Inc
Marvich Maria
McKelvey Terry
LandOfFree
Directed evolution of microorganisms does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Directed evolution of microorganisms, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Directed evolution of microorganisms will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3265967